The ability of human immunodeficiency virus type 1 (HIV-1) to persist in a latent stage in memory T cells in the presence of antiretroviral therapy pcses a major obstacle to the development of an HIV-1 therapy with curative intent. As latently infected cells are phenotypically not distinguishable from uninfected cells, therapeutic reactivation of the latent infection, followed by the death of the host cell induced by viral cytopathicity, is considered the only means to eliminate this viral reservoir. To identify compounds with the potential to reactivate latent HIV-1, we have developed a series of latently HIV-1-infected reporter cell lines that allow for high throughput drug screening (HTS) in a 384-well plate-based format. The latent reporter cell lines use enhanced green fluorescence protein (eGFP) as a direct and quantitative marker of HIV-1 expression. To aid identification of specific compounds, the cells are engineered to constitutively express a second, red fluorescent protein that has no spectral overlap with eGFP, which allows for the simultaneous quantification of cell viability (inversely correlated to compound toxicity). Thus, these reporters enable prioritization of compounds most likely to have a favorable therapeutic window. The high dynamic signal range and the excellent reproducibility of the primary screening assay result in a Z′-factor of 0.89, which characterizes the HTS system as very robust. The assay has been implemented for automated drug screening, and we here discuss the advantages and limitations of the HTS system based on the data obtained for 1,600 compounds during a limited proof-of-concept drug screen. © Mary Ann Liebert, Inc.